A brief review of 8-oxoguanine discussing its mutagenic effects and how its chemical/structural properties affect how it is repaired in DNA
Final Research Project
Henry Liu
Drexel University – Instructor: Jean-Claude Bradley
12/1/2012
8-oxoguanine [Image from ChemSpider page]
CSID: 106574
Contents
Introduction
Reactive Oxygen Species (ROS)
Mutations and DNA Transversions
Mutagenesis of 8-oxoguanine
Structure and Cellular Recognition
Repair Mechanisms and Enzymes
Conclusion
References
Introduction
DNA is the blueprint of life. It is the ultimate source that codes for all the proteins and enzymes responsible for our cellular processes, which in turn directly affect our organ and bodily function for homeostasis and survival. All organisms, including ourselves as human beings, are exposed to an ever-changing environment and are forced to develop adaptations necessary for survival. One such deleterious element that we and our DNA are exposed to are reactive oxygen species (ROS). Reactive oxygen species are capable of modifying normal DNA base pairs causing mutations and transversions to DNA genes [1]. In order to maintain homeostasis, organisms need to find ways to not only prevent exposure to ROS, but also be able to rectify the harmful consequences that could potentially result from unregulated ROS concentrations. The molecule 8-oxoguanine is an oxidatively modified DNA base that has been used to quantify the extent of host DNA damage [2]. Cellular defenses have evolved over the eons throughout both prokaryotes and eukaryotes to be able to deal with DNA damage that can result from ROS or any other mutagenic processes [1]. To be able to further understand how natural enzymes and proteins have evolved to safeguard the DNA from detrimental mutations, it is necessary to examine both the chemical and structural properties of DNA, and the enzymes involved. This paper will discuss the role of the modified base 8-oxoguanine in damaged DNA. Understanding the chemistry of 8-oxoguanine and the role that it plays in DNA replication can help further research in developing preventative measures and treatments against oxidatively damaged host DNA.
Reactive Oxygen Species (ROS)
Reactive oxygen species (ROS) are molecules containing oxygen that are capable of causing undesired chemical reactions within cellular processes. Reactive oxygen species can be found from both endogenous and exogenous sources [2]. ROS are natural byproducts of internal cellular metabolism, which make up the endogenous sources of ROS. Exogenous sources such as ionizing radiation or xenobiotics can also form ROS within the body [3]. In normal cellular metabolism, ROS can be formed from mitochondrial activity as the inner membrane contains multiple electron carriers used in the electron transport chain (ETC) to generate ATP to fuel cellular functions [2]. The electron carriers reduced from the break down of glucose "pass" along electrons to exploit the high energy potential. The final electron acceptor of the chain is an oxygen (aerobic respiration), which is normally supposed to form water upon electron acceptance. However, sometimes it fails to generate water at the end of the chain, and instead the oxygen is incompletely reduced to give the superoxide radical, which is most commonly seen in oxidation-reduction reactions in the (ETC) during NADH dehydrogenase (Complex I) and cytochrome bc1 complex (Complex III) [3]. The superoxide radical anion is biologically toxic due to its high oxidation potential. ROS molecules like superoxide can oxidatively damage integral cellular proteins as well as DNA itself. In addition to direct DNA damage, oxidative damage can inactivate enzymes, cause lipid peroxidation, and disable co-factors for essential metabolic proteins, all of which is capable of inducing apoptosis of the host cell [4]. One of the potential results of oxidative damage include the production of 8-oxoguanine, an oxidatively damaged DNA base [1]. To defend against ROS, cells typically have superoxide "neutralizing" enzyme, such as superoxide dismutase (SOD) [5]. Superoxide dismutases refer to a class of antioxidant enzymes that is capable of catalyzing the reaction that turns superoxide into the less harmful molecules oxygen and hydrogen peroxide. SOD is a widely conserved enzyme among organisms living in environments containing "free" oxygen (such as air) [5]. In humans, there are three forms of SOD that offer antioxidant protection in their respective regions: SOD1 (cytosolic), SOD2 (mitochondrial), SOD3 (extracellular) [5,6].
In 1984, Kasai and Nishimura's studies in hydroxylation of the nucleoside guanine showed that deoxyguanosine is easily oxidized to 8-oxodeoxyguanosine (8-oxodG) by ROS [7]. Their experiment involved hydroxylating guanine residues in DNA at the C-8 position by heated glucose and ROS. Results showed that deoxyguanosine was able to be hydroxylated to form 8-hydroxy-deoxyguanosine (8-OH-dGuo) through various reducing agents, metals, asbestoses, polyphenols, aminophenols, and X-rays [7]. The reactive species for all the methods tested involved an some form of ROS at an intermediate stage. A simple tautomeric shift of 8-hydroxy-deoxyguanosine produces the modified DNA base 8-oxoguanine.
- The hydroxylation and formation of 8-oxoguanine through various reagents. Bottom figure: 8-oxoguanine in equilibrium on the right of 8-hydroxydeoxyguanosine.
dR = deoxyribose sugar in DNA backbone. Kasai H, Nishimura S [7]
Mutations and DNA Transversions
In genetics and the central dogma of molecular biology, DNA is the ultimate root of your physiological make up. In theory, it should possible to prevent all phenotypical shortcomings or diseases, and also become immune to any new strains of viruses or bacteria, with some sort of biogenetic engineering. To create the wide diversity of genetic variability found across the human species, it is important to keep in mind cellular processes such as meiosis and how recombination of chromosome gene segments, law of segregation, and independent assortment in sexual reproduction creates nearly an infinite number of possibilities for genetic variability [8]. Furthermore, a basic grasp of understanding the mechanisms of how genes replicate, transcribe mRNA, and ultimately translate the DNA code for amino acid sequence and protein folding is crucial to understanding how disease and genetic make up affect our everyday lives [8]. But if everything were to always run according to "script", then the annual mortality of humans due to genetic disorders would be nearly nonexistent. Herein lies the inherent "typos" and misprints of of our directions for life, as mutations are introduced into our genetic codes.
A mutation is simply a change of sequence in a coding gene that may or may not affect the phenotypic output of an organism. A mutation can be good or bad, but for the most part, it usually results in adverse effects. This is logical because, evolutionarily through natural selection, we have grown to keep the "desired" genes in the gene pool, while discarding the less favorable ones. So any variation to our genetic make-up (statistically) tends to result in undesired effects. However, an example of a "good" or natural (endogenous) mutation is somatic hypermutation performed by B and T cells of the immune system, to generate a wide variety of antigen-recognizing receptors [8]. Genetic mutations also tend to be spontaneous, occurring only at a low, steady baseline level. But one can certainly increase his or her risk factor for genetic mutations depending on level of exposure to exogenous mutagens (such as radiation or mutagenic viruses) [8]. Ultimately, a change in the DNA sequence can result in altering the product of a gene, or preventing the gene from functioning properly, or it can simply have no effect at all. Mutational changes can fall generally into three categories: point mutations, insertions, and deletions [8]. Point mutations involve several more specific classes, insertions signify the addition of one or more extra base pairs into a DNA sequence, and deletions are the removal of one or more nucleotides from a DNA sequence [8]. We will look more closely at point mutations, and what DNA transversions are.
The most common form of point mutation is the exchange of one of the four base pairs for another, resulting in a erogenous base pair matching when replication or transcription occurs [8]. These types of mutations are further classified as transitions or transversions, with transitions being the more common type [9]. Transition point mutations involve the switch between nucleotides of the same heterocyclic ring member families: purines (adenine and guanine) are switched with another purine, and pyrimidines (thymine and cytosine) are switch with another pyrimidine [8]. Transversions are the opposite: purines are switch with pyrimidines, and vice versa. Although transversions are less common, their downstream effects can be more dramatic than transition point mutations. This is due to the fact that the chemical structures of purines (which are heterocyclic aromatics with two rings conjoined) and pyrimidines (which is a single heterocyclic ring) differ spatially, which can result in more miscoding during protein translation by ribosomes [9]. 8-oxoguanine introduces a DNA lesion that results from a G:C->T:A transversion point mutation, which is the root of the mutagenic effects of 8-oxoguanine [10].
Mutagenesis of 8-oxoguanine
As mentioned previously, deoxyguanosine is easily oxidized to 8-oxodeoxyguanosine (8-oxodG). About 1/100,000 guanine resides in human DNA is oxidized at the C-8 carbon position leading to endogenous DNA "miscoding" [9]. 8-oxodG essentially creates lesions in DNA replication or transcription either halting the process, or creating a misincorporated base pair, leading to transversion mutations in genes. 8-oxodG miscodes by incorporating the dNMPs deoxyadenosine monophosphate (dAMP) and deoxycytidine monophosphate (dCMP) opposite 8-oxodG in vitro [10]. DNA replication polymerases pol [alpha], pol [delta] and pol III incorporate dAMP opposite 8-oxodG, while DNA replication polymerases pol I and pol [beta] incorporates dCMP opposite 8-oxodG. Mutagenic potentials are generally protected against by exo-nucleolytic proofreading [9]. However, the 3'->5' exo-nucleolytic proofreading of pol I and pol III does not fix the incorrect 8-oxodG:dAMP pairing. Usually, incorrectly paired damaged bases generally are not extended efficiently in DNA polymerases, however 8-oxodG:dA is somewhat of an exception [9]. The DNA strand (either the newly synthesized or the template strand) downstream of the 8-oxodG:dA mismatch pairing is capable of extending from the 3' primer terminus at half the rate of normal G:C or A:T pairs. Structural analysis show that the 8-oxodG(syn):dA(anti) conformation of the base pair shields the 3' end from exonucleolytic proofreading [9-10].
However, maybe the mutagenic potential of 8-oxodG isn't as bad as it sounds. Despite the fact that this mismatch pairing leading to mutational transversion in DNA is poorly corrected by pol I and pol III, research results show that the harmful mutagenic effects aren't as prevalent as they're spelled out to be [11]. Studies were performed with plasmid vectors containing a single lesion to determine the mutational frequency and specificity of 8-oxodG. The majority of mutations produced by the 8-oxodG:dA mutation were G:C->T:A transversions. The findings of the studies show that mutational frequencies throughout all the trials were <5%, revealing 8-oxodG to be weakly mutagenic in vivo [9]. While the presence of 8-oxodG did not greatly affect the number of mutations, studies performed by M. Moriya et al (1991) showed that a significant number of G:C->A:T transversions were observed in the absence of SOS cell induction [12]. The SOS cellular response is exactly as it sounds: an emergency state of the host cell in dire need of rescue. It is characterized by a state of high-activity DNA repair, and can be induced by exposure to DNA-damaging agents. DNA at the site of damage is lesioned and quickly fragmented into components and primitive repair mechanisms are deployed to patch whatever gene segments it can save, even at the cost of error-prone base pair incorporations. This suggests that intranuclear preventative mechanisms play a crucial role in monitoring for the mutagenic potential of this type of lesion [10].
Structure and Cellular Recognition
Deoxyguanosine forms two conformational configurations when bound to the DNA sugar backbone about the C-8 carbon: 8-oxodG(syn) and 8-oxodG(anti). The (syn) and (anti) forms of 8-oxodG form an rapidly shifting equilibrium, but with the syn conformation being energetically preferred [10]. The conformation of 8-oxodG is important because it can play a determining factor deciding which base it is paired with, when recognized by DNA polymerases. Because dGTP can be oxidized by ROS, 8-oxodGTP is present in the dNTP pool [12]. DNA polymerases pair 8-oxodGMP with dC or dA on the template DNA strand, forming A:T->C:G transversions. When 8-oxodGTP is bound to DNA polymerase, the equilibrium between the two conformations may be changed; in the (syn) conformation, it is paired with dA on the synthesized strand, and in the (anti) conformation it is likely paired with dC [10]. Likewise, 8-oxodG positioned in the template strand may assume syn or anti conformations. Further NMR studies suggest that, depending on the dNMP inserted, steric repulsion between the 8-oxo and deoxyribose surfaces in the 8-oxodG(anti):dC(anti) conformation is stabilized by a third hydrogen bond, showing that the (anti) conformation is preferential to dC pairing by DNA polymerase [13].
As mentioned before, the 8-oxodG(syn): dA(anti) conformation is not easily recognized and corrected by proofreading mechanisms. Because of this, formation of the 8-oxodG(syn):dA(anti) is predominantly the reason for mutagenesis during DNA replication [13]. The steric positions of the hydrogen bonds in the major and minor grooves of the DNA affects its recognition by DNA polymerases, which affects binding and incision by DNA glycosylases during DNA repair. Three DNA structural conformation combinations can be considered: 8-oxodG(syn) opposite dA(anti), 8-oxodG(anti) opposite dC(anti), and 8-oxodG(syn) opposite an abasic site [13]. An abasic site, or AP site (apurinic/apyrimidinic site), is a part of a sequence in the DNA strand that does not contain a typical purine or a pyrimidine base, generally due to DNA damage. The first two structures represent natural substrates for 8-oxoguanine DNA glycosylase and adenine DNA glycosylase, respectively; the third is an intermediate in 8-oxodG repair [14]. When 8-oxodG is anti, the carbonyl group at C-8 resides in the major groove and the exocyclic amino group is found in the minor groove. In theory, DNA glycosylase could distinguish between two 8-oxopurines by binding the exocyclic amino group of 8-oxodG; however, the enzyme is required to make contact with DNA structure in both the minor and major grooves [14]. Further structural analysis with NMR techniques by Krahn, J. et al (2003) shows that a more plausible hypothesis for enzyme-substrate recognition involves stereochemistry about the C-6 and C-8 carbonyl of 8-oxodG [15].
- Conformationally preferred forms of 8-oxodG base pairs. Krahn JM, Beard WA, Miller H, Grollman AP, Wilson SH [15]
While 8-oxodG(syn):dA(anti) serves as a non-binding substrate to 8-oxoguanine DNA glycosylase, it is the naturally occuring substrate for the enzyme adenine DNA glycosylase [16]. Adenine glycosylase inhibits 8-oxoguanine DNA glycosylase, suggesting that both enzymes compete for the same binding site. DNA glycosylases are enzymes involved in base excision repair, removal, and repair [14]. However, it was somewhat surprising to find that the 8-oxo group is not involved in the DNA site recognition process, the very site that marks it as oxidative damage [16]. Presumably adenine DNA glycosylase encounters hydrogen bonds common to guanine(syn) and 8-oxoguanine(syn), and engages recognition with the amino functional group of dA(anti) [17]. Further structural analysis will be required to determine the relationship between the binding of DNA glycosylase and the repair effects of 8-oxoguanine.
Repair Mechanisms and Enzymes
In 1992, Michaels and Miller performed experiments with different strains of E. coli strains that produced different repair proteins in situations of cellular stress or significant DNA damage [18]. The glycosylase enzymes involved were specific to repairing 8-oxoguanine DNA lesions, which was a general error avoidance pathway that was meant to enhance the fidelity of replicating DNA strands [18]. It has also been proven that these enzymes involved are not only conserved throughout other prokaryotes, but also higher eukaryotes, such as humans (we contain an analogue of these repair enzymes). The E. coli system of repair enzymes involve at least three different strains of genes, MutM, MutT, and MutY [19]. These three enzymes are responsible for fixing oxidatively damaged forms of guanine nucleotides from the DNA and correcting error-prone lesion in DNA synthesis. Based on research for these three proteins, extrapolations can be made about human analogue protein equivalents, but more on this later. Additionally, Tchou, J. et al (1993) found that 8-oxoguanine-DNA glycosylase (Fpg protein) of E. coli has N-glycosylase and apurinic/apyrimidinic (AP) lyase functions during DNA replication to ensure fidelity [19]. This enzyme fixes oxidative damage by removing the lesions and 8-oxoguanine misinsorporations from the synthesized strand. In their research, the Fpg protein was used to determine the structure of various binding sites on the template strand, based on binding affinities of Fpg to damaged lesions or point mutations [19]. These were all the sites that were recognizable by the repair enzymes, and failure or inefficiency of these enzymes resulted in higher incidences in mutations
It is imperative that all living organisms must develop some sort of defense and repair mechanisms to prevent the mutagenic effects of any type of DNA lesion, regardless if it caused by 8-oxoguanine or not [20]. 8-oxodGTP pairs with almost equal efficiency to dC and dA, generating a high frequency of A:T->C:G transversions. This presents us with the first level of protection: a nucleotide triphosphatase that hydrolyses 8-oxodGTP, preventing it from being incorporated in any proliferating DNA strands. This effectively removes 8-oxodGTP from the nucleotide pool and prevents its insertion in the coding gene [20]. A second level of protection involves 8-oxoguanine DNA glycosylase mentioned before (formamidopyrimidine DNA glycosylase or Fpg protein). This enzyme removes 8-oxoguanine from DNA where the lesion is paired with dC, repairing the base pair [21]. The glycosylase is less active on 8-oxodG:dA pairing, which is the favored pairing during DNA replication. 8-oxoguanine DNA glycosylase defends against mutagenesis by promoting G:C->T:A transversions [21]. A third level of protection occurs when 8-oxodG is failed to be repaired by 8-oxoguanine DNA glycosylase. Downstream enzymatic removal repairs the misincorporated adenine, but full repair of the lesion is not effected until dCMP (the correct base pair match) is inserted in the lesioned DNA strand [22].
For the different strains of E. coli mentioned earlier, there are consequences that arise of the different levels of protection. MutT strains are deficient in adenine DNA glycosylase and have G:C->T:A mutations [21]. The mutT overproduces 8-oxoguanine DNA glycosylase, suggesting that 8-oxodG:dA is the natural substrate for this enzyme. However, studies seem to show that a mutM and mutY double mutation seem to work together to protect against G:C->T:A mutations, suggesting evidence that shows the mutY gene in E. coli produce an enzyme that directly reduce the frequency of G:C->T:A mutations in vivo [22]. Misincorporated adenine residues are removed by adenine DNA glycosylase, creating an abasic site, which is incised by apurinic/apyrimidinic (AP) endonuclease activity and the gap is filled by pol I to pair dCMP opposite 8-oxodG. This produces the substrate for 8-oxoguanine DNA glycosylase, which has the ability to ultimately restore the natural base pairing [23]. Finally, mammalian counterparts of the products of the E. coli genes mutM, mutY and mutT have been identified and it appears that an analogous defense system operates in mammalian cells, suggesting that our repair mechanisms that keep the mutagenic effects of 8-oxoguanine at a minimum [24].
Conclusion
As living organisms, we are all forced to adapt, thrive and survive in this ever-changing environment. Sometimes we are very fortunate and are able to take advantage of the plentiful resources and opportunities that are presented to us without much hardship or challenges. But other times (more often than not), we have to learn and change our ways to overcome those hardships or challenges that come across our path. This general rule applies even to the biochemistry of our cellular metabolism. Mandatory oxygen that is necessary for survival is capable of producing toxic metabolites called reactive oxygen species (ROS) that can damage the integrity of DNA and cellular functions [2]. The deleterious oxidative effects of 8-oxoguanine in replicating DNA is combated by enzymes such as superoxide dismutase (SOD), which reduces the levels of cellular ROS, either from exogenous or endogenous sources [5]. Misincorporated 8-oxoguanine base pairs in the DNA causes lesions in coding segments that results in mutagenic effects. These effects are dampened and defended against by nucleotide triphosphatases as well as DNA glycosylases such as the Fpg protein and AP endonucleases [15]. Studies have been performed with different strands of E. coli, but the results can be interpolated into human analogues involving the same repair enzymes and mechanisms [19]. Further research would be needed to demonstrate the efficiency of repair enzymes under different conditions such as cellular stress of immunological infections, but the research performed thus far show promising effects of endogenous defense mechanisms that protect against a wide variety of DNA damage.
References
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Kasai H, Nishimura S. Hydroxylation of guanine in nucleosides and DNA at the C-8 position by heated glucose and oxygen radical-forming agents. Environmental Health Perspectives. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1474402/> Accessed December 1 2012. 1986;67:111-116.
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Moriya M, Ou C, Bodepudi V, Johnson F, Takeshita M, Grollman A. Site-specific mutagenesis using a gapped duplex vector: A study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutation Research/DNA Repair. 1991;254(3):281-288. DOI: 10.1016/0921-8777(91)90067-Y
Kouchakdjian M, Bodepudi V, Shibutani S, Eisenberg M, Johnson F, Grollman AP, Patel DJ. NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-oxo-7H-dG(syn).cntdot.dA(anti) alignment at lesion site. Biochemistry (NY). 1991;30(5):1403-1412. DOI: 10.1021/bi00219a034
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Link to Henry Liu-Log
A brief review of 8-oxoguanine discussing its mutagenic effects and how its chemical/structural properties affect how it is repaired in DNA
Final Research ProjectHenry Liu
Drexel University – Instructor: Jean-Claude Bradley
12/1/2012
8-oxoguanine [Image from ChemSpider page]
CSID: 106574
Contents
Introduction
Reactive Oxygen Species (ROS)
Mutations and DNA Transversions
Mutagenesis of 8-oxoguanine
Structure and Cellular Recognition
Repair Mechanisms and Enzymes
Conclusion
References
Introduction
DNA is the blueprint of life. It is the ultimate source that codes for all the proteins and enzymes responsible for our cellular processes, which in turn directly affect our organ and bodily function for homeostasis and survival. All organisms, including ourselves as human beings, are exposed to an ever-changing environment and are forced to develop adaptations necessary for survival. One such deleterious element that we and our DNA are exposed to are reactive oxygen species (ROS). Reactive oxygen species are capable of modifying normal DNA base pairs causing mutations and transversions to DNA genes [1]. In order to maintain homeostasis, organisms need to find ways to not only prevent exposure to ROS, but also be able to rectify the harmful consequences that could potentially result from unregulated ROS concentrations. The molecule 8-oxoguanine is an oxidatively modified DNA base that has been used to quantify the extent of host DNA damage [2]. Cellular defenses have evolved over the eons throughout both prokaryotes and eukaryotes to be able to deal with DNA damage that can result from ROS or any other mutagenic processes [1]. To be able to further understand how natural enzymes and proteins have evolved to safeguard the DNA from detrimental mutations, it is necessary to examine both the chemical and structural properties of DNA, and the enzymes involved. This paper will discuss the role of the modified base 8-oxoguanine in damaged DNA. Understanding the chemistry of 8-oxoguanine and the role that it plays in DNA replication can help further research in developing preventative measures and treatments against oxidatively damaged host DNA.
Reactive Oxygen Species (ROS)
Reactive oxygen species (ROS) are molecules containing oxygen that are capable of causing undesired chemical reactions within cellular processes. Reactive oxygen species can be found from both endogenous and exogenous sources [2]. ROS are natural byproducts of internal cellular metabolism, which make up the endogenous sources of ROS. Exogenous sources such as ionizing radiation or xenobiotics can also form ROS within the body [3]. In normal cellular metabolism, ROS can be formed from mitochondrial activity as the inner membrane contains multiple electron carriers used in the electron transport chain (ETC) to generate ATP to fuel cellular functions [2]. The electron carriers reduced from the break down of glucose "pass" along electrons to exploit the high energy potential. The final electron acceptor of the chain is an oxygen (aerobic respiration), which is normally supposed to form water upon electron acceptance. However, sometimes it fails to generate water at the end of the chain, and instead the oxygen is incompletely reduced to give the superoxide radical, which is most commonly seen in oxidation-reduction reactions in the (ETC) during NADH dehydrogenase (Complex I) and cytochrome bc1 complex (Complex III) [3]. The superoxide radical anion is biologically toxic due to its high oxidation potential. ROS molecules like superoxide can oxidatively damage integral cellular proteins as well as DNA itself. In addition to direct DNA damage, oxidative damage can inactivate enzymes, cause lipid peroxidation, and disable co-factors for essential metabolic proteins, all of which is capable of inducing apoptosis of the host cell [4]. One of the potential results of oxidative damage include the production of 8-oxoguanine, an oxidatively damaged DNA base [1]. To defend against ROS, cells typically have superoxide "neutralizing" enzyme, such as superoxide dismutase (SOD) [5]. Superoxide dismutases refer to a class of antioxidant enzymes that is capable of catalyzing the reaction that turns superoxide into the less harmful molecules oxygen and hydrogen peroxide. SOD is a widely conserved enzyme among organisms living in environments containing "free" oxygen (such as air) [5]. In humans, there are three forms of SOD that offer antioxidant protection in their respective regions: SOD1 (cytosolic), SOD2 (mitochondrial), SOD3 (extracellular) [5,6].
In 1984, Kasai and Nishimura's studies in hydroxylation of the nucleoside guanine showed that deoxyguanosine is easily oxidized to 8-oxodeoxyguanosine (8-oxodG) by ROS [7]. Their experiment involved hydroxylating guanine residues in DNA at the C-8 position by heated glucose and ROS. Results showed that deoxyguanosine was able to be hydroxylated to form 8-hydroxy-deoxyguanosine (8-OH-dGuo) through various reducing agents, metals, asbestoses, polyphenols, aminophenols, and X-rays [7]. The reactive species for all the methods tested involved an some form of ROS at an intermediate stage. A simple tautomeric shift of 8-hydroxy-deoxyguanosine produces the modified DNA base 8-oxoguanine.
- The hydroxylation and formation of 8-oxoguanine through various reagents. Bottom figure: 8-oxoguanine in equilibrium on the right of 8-hydroxydeoxyguanosine.
dR = deoxyribose sugar in DNA backbone. Kasai H, Nishimura S [7]
Mutations and DNA Transversions
In genetics and the central dogma of molecular biology, DNA is the ultimate root of your physiological make up. In theory, it should possible to prevent all phenotypical shortcomings or diseases, and also become immune to any new strains of viruses or bacteria, with some sort of biogenetic engineering. To create the wide diversity of genetic variability found across the human species, it is important to keep in mind cellular processes such as meiosis and how recombination of chromosome gene segments, law of segregation, and independent assortment in sexual reproduction creates nearly an infinite number of possibilities for genetic variability [8]. Furthermore, a basic grasp of understanding the mechanisms of how genes replicate, transcribe mRNA, and ultimately translate the DNA code for amino acid sequence and protein folding is crucial to understanding how disease and genetic make up affect our everyday lives [8]. But if everything were to always run according to "script", then the annual mortality of humans due to genetic disorders would be nearly nonexistent. Herein lies the inherent "typos" and misprints of of our directions for life, as mutations are introduced into our genetic codes.
A mutation is simply a change of sequence in a coding gene that may or may not affect the phenotypic output of an organism. A mutation can be good or bad, but for the most part, it usually results in adverse effects. This is logical because, evolutionarily through natural selection, we have grown to keep the "desired" genes in the gene pool, while discarding the less favorable ones. So any variation to our genetic make-up (statistically) tends to result in undesired effects. However, an example of a "good" or natural (endogenous) mutation is somatic hypermutation performed by B and T cells of the immune system, to generate a wide variety of antigen-recognizing receptors [8]. Genetic mutations also tend to be spontaneous, occurring only at a low, steady baseline level. But one can certainly increase his or her risk factor for genetic mutations depending on level of exposure to exogenous mutagens (such as radiation or mutagenic viruses) [8]. Ultimately, a change in the DNA sequence can result in altering the product of a gene, or preventing the gene from functioning properly, or it can simply have no effect at all. Mutational changes can fall generally into three categories: point mutations, insertions, and deletions [8]. Point mutations involve several more specific classes, insertions signify the addition of one or more extra base pairs into a DNA sequence, and deletions are the removal of one or more nucleotides from a DNA sequence [8]. We will look more closely at point mutations, and what DNA transversions are.
The most common form of point mutation is the exchange of one of the four base pairs for another, resulting in a erogenous base pair matching when replication or transcription occurs [8]. These types of mutations are further classified as transitions or transversions, with transitions being the more common type [9]. Transition point mutations involve the switch between nucleotides of the same heterocyclic ring member families: purines (adenine and guanine) are switched with another purine, and pyrimidines (thymine and cytosine) are switch with another pyrimidine [8]. Transversions are the opposite: purines are switch with pyrimidines, and vice versa. Although transversions are less common, their downstream effects can be more dramatic than transition point mutations. This is due to the fact that the chemical structures of purines (which are heterocyclic aromatics with two rings conjoined) and pyrimidines (which is a single heterocyclic ring) differ spatially, which can result in more miscoding during protein translation by ribosomes [9]. 8-oxoguanine introduces a DNA lesion that results from a G:C->T:A transversion point mutation, which is the root of the mutagenic effects of 8-oxoguanine [10].
Mutagenesis of 8-oxoguanine
As mentioned previously, deoxyguanosine is easily oxidized to 8-oxodeoxyguanosine (8-oxodG). About 1/100,000 guanine resides in human DNA is oxidized at the C-8 carbon position leading to endogenous DNA "miscoding" [9]. 8-oxodG essentially creates lesions in DNA replication or transcription either halting the process, or creating a misincorporated base pair, leading to transversion mutations in genes. 8-oxodG miscodes by incorporating the dNMPs deoxyadenosine monophosphate (dAMP) and deoxycytidine monophosphate (dCMP) opposite 8-oxodG in vitro [10]. DNA replication polymerases pol [alpha], pol [delta] and pol III incorporate dAMP opposite 8-oxodG, while DNA replication polymerases pol I and pol [beta] incorporates dCMP opposite 8-oxodG. Mutagenic potentials are generally protected against by exo-nucleolytic proofreading [9]. However, the 3'->5' exo-nucleolytic proofreading of pol I and pol III does not fix the incorrect 8-oxodG:dAMP pairing. Usually, incorrectly paired damaged bases generally are not extended efficiently in DNA polymerases, however 8-oxodG:dA is somewhat of an exception [9]. The DNA strand (either the newly synthesized or the template strand) downstream of the 8-oxodG:dA mismatch pairing is capable of extending from the 3' primer terminus at half the rate of normal G:C or A:T pairs. Structural analysis show that the 8-oxodG(syn):dA(anti) conformation of the base pair shields the 3' end from exonucleolytic proofreading [9-10].
However, maybe the mutagenic potential of 8-oxodG isn't as bad as it sounds. Despite the fact that this mismatch pairing leading to mutational transversion in DNA is poorly corrected by pol I and pol III, research results show that the harmful mutagenic effects aren't as prevalent as they're spelled out to be [11]. Studies were performed with plasmid vectors containing a single lesion to determine the mutational frequency and specificity of 8-oxodG. The majority of mutations produced by the 8-oxodG:dA mutation were G:C->T:A transversions. The findings of the studies show that mutational frequencies throughout all the trials were <5%, revealing 8-oxodG to be weakly mutagenic in vivo [9]. While the presence of 8-oxodG did not greatly affect the number of mutations, studies performed by M. Moriya et al (1991) showed that a significant number of G:C->A:T transversions were observed in the absence of SOS cell induction [12]. The SOS cellular response is exactly as it sounds: an emergency state of the host cell in dire need of rescue. It is characterized by a state of high-activity DNA repair, and can be induced by exposure to DNA-damaging agents. DNA at the site of damage is lesioned and quickly fragmented into components and primitive repair mechanisms are deployed to patch whatever gene segments it can save, even at the cost of error-prone base pair incorporations. This suggests that intranuclear preventative mechanisms play a crucial role in monitoring for the mutagenic potential of this type of lesion [10].
Structure and Cellular Recognition
Deoxyguanosine forms two conformational configurations when bound to the DNA sugar backbone about the C-8 carbon: 8-oxodG(syn) and 8-oxodG(anti). The (syn) and (anti) forms of 8-oxodG form an rapidly shifting equilibrium, but with the syn conformation being energetically preferred [10]. The conformation of 8-oxodG is important because it can play a determining factor deciding which base it is paired with, when recognized by DNA polymerases. Because dGTP can be oxidized by ROS, 8-oxodGTP is present in the dNTP pool [12]. DNA polymerases pair 8-oxodGMP with dC or dA on the template DNA strand, forming A:T->C:G transversions. When 8-oxodGTP is bound to DNA polymerase, the equilibrium between the two conformations may be changed; in the (syn) conformation, it is paired with dA on the synthesized strand, and in the (anti) conformation it is likely paired with dC [10]. Likewise, 8-oxodG positioned in the template strand may assume syn or anti conformations. Further NMR studies suggest that, depending on the dNMP inserted, steric repulsion between the 8-oxo and deoxyribose surfaces in the 8-oxodG(anti):dC(anti) conformation is stabilized by a third hydrogen bond, showing that the (anti) conformation is preferential to dC pairing by DNA polymerase [13].
As mentioned before, the 8-oxodG(syn): dA(anti) conformation is not easily recognized and corrected by proofreading mechanisms. Because of this, formation of the 8-oxodG(syn):dA(anti) is predominantly the reason for mutagenesis during DNA replication [13]. The steric positions of the hydrogen bonds in the major and minor grooves of the DNA affects its recognition by DNA polymerases, which affects binding and incision by DNA glycosylases during DNA repair. Three DNA structural conformation combinations can be considered: 8-oxodG(syn) opposite dA(anti), 8-oxodG(anti) opposite dC(anti), and 8-oxodG(syn) opposite an abasic site [13]. An abasic site, or AP site (apurinic/apyrimidinic site), is a part of a sequence in the DNA strand that does not contain a typical purine or a pyrimidine base, generally due to DNA damage. The first two structures represent natural substrates for 8-oxoguanine DNA glycosylase and adenine DNA glycosylase, respectively; the third is an intermediate in 8-oxodG repair [14]. When 8-oxodG is anti, the carbonyl group at C-8 resides in the major groove and the exocyclic amino group is found in the minor groove. In theory, DNA glycosylase could distinguish between two 8-oxopurines by binding the exocyclic amino group of 8-oxodG; however, the enzyme is required to make contact with DNA structure in both the minor and major grooves [14]. Further structural analysis with NMR techniques by Krahn, J. et al (2003) shows that a more plausible hypothesis for enzyme-substrate recognition involves stereochemistry about the C-6 and C-8 carbonyl of 8-oxodG [15].
- Conformationally preferred forms of 8-oxodG base pairs. Krahn JM, Beard WA, Miller H, Grollman AP, Wilson SH [15]
While 8-oxodG(syn):dA(anti) serves as a non-binding substrate to 8-oxoguanine DNA glycosylase, it is the naturally occuring substrate for the enzyme adenine DNA glycosylase [16]. Adenine glycosylase inhibits 8-oxoguanine DNA glycosylase, suggesting that both enzymes compete for the same binding site. DNA glycosylases are enzymes involved in base excision repair, removal, and repair [14]. However, it was somewhat surprising to find that the 8-oxo group is not involved in the DNA site recognition process, the very site that marks it as oxidative damage [16]. Presumably adenine DNA glycosylase encounters hydrogen bonds common to guanine(syn) and 8-oxoguanine(syn), and engages recognition with the amino functional group of dA(anti) [17]. Further structural analysis will be required to determine the relationship between the binding of DNA glycosylase and the repair effects of 8-oxoguanine.
Repair Mechanisms and Enzymes
In 1992, Michaels and Miller performed experiments with different strains of E. coli strains that produced different repair proteins in situations of cellular stress or significant DNA damage [18]. The glycosylase enzymes involved were specific to repairing 8-oxoguanine DNA lesions, which was a general error avoidance pathway that was meant to enhance the fidelity of replicating DNA strands [18]. It has also been proven that these enzymes involved are not only conserved throughout other prokaryotes, but also higher eukaryotes, such as humans (we contain an analogue of these repair enzymes). The E. coli system of repair enzymes involve at least three different strains of genes, MutM, MutT, and MutY [19]. These three enzymes are responsible for fixing oxidatively damaged forms of guanine nucleotides from the DNA and correcting error-prone lesion in DNA synthesis. Based on research for these three proteins, extrapolations can be made about human analogue protein equivalents, but more on this later. Additionally, Tchou, J. et al (1993) found that 8-oxoguanine-DNA glycosylase (Fpg protein) of E. coli has N-glycosylase and apurinic/apyrimidinic (AP) lyase functions during DNA replication to ensure fidelity [19]. This enzyme fixes oxidative damage by removing the lesions and 8-oxoguanine misinsorporations from the synthesized strand. In their research, the Fpg protein was used to determine the structure of various binding sites on the template strand, based on binding affinities of Fpg to damaged lesions or point mutations [19]. These were all the sites that were recognizable by the repair enzymes, and failure or inefficiency of these enzymes resulted in higher incidences in mutations
It is imperative that all living organisms must develop some sort of defense and repair mechanisms to prevent the mutagenic effects of any type of DNA lesion, regardless if it caused by 8-oxoguanine or not [20]. 8-oxodGTP pairs with almost equal efficiency to dC and dA, generating a high frequency of A:T->C:G transversions. This presents us with the first level of protection: a nucleotide triphosphatase that hydrolyses 8-oxodGTP, preventing it from being incorporated in any proliferating DNA strands. This effectively removes 8-oxodGTP from the nucleotide pool and prevents its insertion in the coding gene [20]. A second level of protection involves 8-oxoguanine DNA glycosylase mentioned before (formamidopyrimidine DNA glycosylase or Fpg protein). This enzyme removes 8-oxoguanine from DNA where the lesion is paired with dC, repairing the base pair [21]. The glycosylase is less active on 8-oxodG:dA pairing, which is the favored pairing during DNA replication. 8-oxoguanine DNA glycosylase defends against mutagenesis by promoting G:C->T:A transversions [21]. A third level of protection occurs when 8-oxodG is failed to be repaired by 8-oxoguanine DNA glycosylase. Downstream enzymatic removal repairs the misincorporated adenine, but full repair of the lesion is not effected until dCMP (the correct base pair match) is inserted in the lesioned DNA strand [22].
For the different strains of E. coli mentioned earlier, there are consequences that arise of the different levels of protection. MutT strains are deficient in adenine DNA glycosylase and have G:C->T:A mutations [21]. The mutT overproduces 8-oxoguanine DNA glycosylase, suggesting that 8-oxodG:dA is the natural substrate for this enzyme. However, studies seem to show that a mutM and mutY double mutation seem to work together to protect against G:C->T:A mutations, suggesting evidence that shows the mutY gene in E. coli produce an enzyme that directly reduce the frequency of G:C->T:A mutations in vivo [22]. Misincorporated adenine residues are removed by adenine DNA glycosylase, creating an abasic site, which is incised by apurinic/apyrimidinic (AP) endonuclease activity and the gap is filled by pol I to pair dCMP opposite 8-oxodG. This produces the substrate for 8-oxoguanine DNA glycosylase, which has the ability to ultimately restore the natural base pairing [23]. Finally, mammalian counterparts of the products of the E. coli genes mutM, mutY and mutT have been identified and it appears that an analogous defense system operates in mammalian cells, suggesting that our repair mechanisms that keep the mutagenic effects of 8-oxoguanine at a minimum [24].
Conclusion
As living organisms, we are all forced to adapt, thrive and survive in this ever-changing environment. Sometimes we are very fortunate and are able to take advantage of the plentiful resources and opportunities that are presented to us without much hardship or challenges. But other times (more often than not), we have to learn and change our ways to overcome those hardships or challenges that come across our path. This general rule applies even to the biochemistry of our cellular metabolism. Mandatory oxygen that is necessary for survival is capable of producing toxic metabolites called reactive oxygen species (ROS) that can damage the integrity of DNA and cellular functions [2]. The deleterious oxidative effects of 8-oxoguanine in replicating DNA is combated by enzymes such as superoxide dismutase (SOD), which reduces the levels of cellular ROS, either from exogenous or endogenous sources [5]. Misincorporated 8-oxoguanine base pairs in the DNA causes lesions in coding segments that results in mutagenic effects. These effects are dampened and defended against by nucleotide triphosphatases as well as DNA glycosylases such as the Fpg protein and AP endonucleases [15]. Studies have been performed with different strands of E. coli, but the results can be interpolated into human analogues involving the same repair enzymes and mechanisms [19]. Further research would be needed to demonstrate the efficiency of repair enzymes under different conditions such as cellular stress of immunological infections, but the research performed thus far show promising effects of endogenous defense mechanisms that protect against a wide variety of DNA damage.
References